Breast Tumor Biopsies Research Articles

Abstract 5576: Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening

Abstract Screening and prevention is critical to reducing breast cancer deaths, and mammography is not an ideal tool. Mammography has not reduced invasive breast cancer incidence, it has high numbers of false positives and it detects many non-invasive cancers that are not life threatening. Metastatic breast cancer is the second leading cause of cancer death for women. The development of a new blood test for breast cancer screening could offer significant improvements with the potential to be more accurate, less costly, less intimidating, not subject to age differences, and able to discriminate between cancers subtypes. Detection of breast cancer cells (BCC) in blood samples offers a valuable opportunity for screening. However, BCC in blood are rare and must be identified against a background of millions of other blood cells. Although many technologies have been developed for the isolation and detection of circulating tumor cells using EpCAM and cytokeratins, these tests do not provide levels of sensitivity and specificity required for a cancer screening tool. To address this critical unmet need, we developed a panel of cell surface markers whose expression patterns specifically distinguish breast cancer cells in blood samples. These genes were selected based on a discovery platform tailored to detect early stage breast cancers and include 20 novel putative cell surface molecules that are candidates for breast cancer cell detection and isolation. We prioritized these molecules by specificity of expression in tumor cells including: 1) Ratio >25:1 average breast tumor biopsy expression versus average normal blood expression, 2) High expression relative (>3 fold) in >20% of tumor biopsy samples, 3) High expression (>3 fold) in >40% of breast cancer cell lines, and 4) Expression significantly associated with 10-year outcomes (Kaplan Meier analysis, p<0.05). We combined the top 10 ranking genes in each of these categories, resulting in a list of 20 candidates that represent high priority targets. Antibodies for all 20 genes are commercially available. Expression in micro dissected biopsies was markedly higher than non-breast cancer blood for most. Also, most were expressed at lower levels than EpCAM in normal breast tissue and had background level expression in isolated normal blood cells. The innovation of this strategy is twofold. First, it uses a panel of molecules instead of an individual molecule to detect and isolate breast cancer cells and, second, it relies on molecules that are clinically relevant to breast cancers, rather than relying on generic epithelial cell markers. Current studies include the validation of antibody cocktails with specificity and sensitivity that outperforms the current EpCAM enrichment method and provides a proof of feasibility for screening of breast cancer. Citation Format: Lucas Delmonico, Edward C. Goodwin, Marcia Vasconcellos Fournier. Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5576.

Role of Farnesoid X Receptor Activation in Counteracting Tumor‐promoting Functions of Cancer‐Associated Fibroblasts in Breast Cancers

Cancer‐associated fibroblasts (CAFs), the principal components of the tumor microenvironment, play multiple roles in breast cancer onset and progression. While their significance is widely accepted, treatment options to target CAFs in clinical practice have not yet been well established. The nuclear receptor superfamily encompasses a druggable class of molecules, expressed in various stroma and parenchymal cell types, with the interesting therapeutic potential to modulate also the reactive microenvironment. Previously, we have demonstrated that activation of the nuclear Farnesoid X Receptor (FXR) in mammary epithelial cancer cells was associated with a reduction of cell proliferation, migration, and invasion ‘in vitro’ and tumor growth ‘in vivo’. To extend these observations, here, we propose to assess the function of FXR in CAFs and evaluate whether this receptor may affect their tumor‐promoting features.Our results evidenced, for the first time, FXR mRNA and protein expression in CAFs isolated from biopsies of primary human breast tumors. Treatment with the FXR agonist GW4064 treatment was able to significantly decrease CAF motility, stress fiber formation and their contractility. RNA sequencing highlighted cell movement and migration of cells among the most down‐regulated functions and identified signalings known to govern cell cytoskeleton organization and migration (i.e. ILK‐actin cytoskeletal‐Rho signalings) among the most down‐regulated ingenuity canonical pathways in response to GW4064 treatment. Accordingly, decreased expression and activation of proteins and/or downstream effectors of these pathways were observed in GW4064‐treated CAFs. Moreover, FXR activation reduced expression of different secreted factors, that act as mediators of tumor‐stroma interaction. Indeed, coculture experiments revealed a significant inhibition in growth and motility of breast cancer cells treated with conditioned‐media derived from GW4064‐treated CAFs compared to vehicle‐treated CAFs. All this sounds to fit with the clinical relevance that increased FXR levels in bulk tumors are associated with a statistically significant longer survival of patients.In conclusion, FXR ligands, by targeting both breast cancer epithelial cells and tumor associated stroma, may represent emerging pharmacological tools to be exploited for the future management of breast cancer.Support or Funding InformationMy First AIRC Grant #16899 to IBThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated.

The Proliferative Response to p27 Down-Regulation in Estrogen Plus Progestin Hormonal Therapy is Lost in Breast Tumors

Increased proliferation and breast cancer risk has been observed in postmenopausal women receiving estrogen (E) + progestin hormone replacement therapy (HRT). Progestin action is mediated through two progesterone receptor (PR) isoforms, PRA and PRB, with unique transcriptional activity and function. The current study examines hormonal regulation of PR isoforms in the normal postmenopausal human breast and the mechanism by which progestins increase proliferation and breast cancer risk. Archival benign breast biopsies from postmenopausal and premenopausal women, and luminal breast tumor biopsies from postmenopausal women, were analyzed for regulation of PRA and PRB expression by E and E+medroxyprogesterone acetate (MPA). In the postmenopausal breast without HRT, PRA and PRB expression was decreased compared to the premenopausal breast. Both E (n = 12) and E+MPA (n = 13) HRT in the postmenopausal breast were associated with increased PRA and PRB expression, increased nuclear cyclin E expression, and decreased nuclear p27 expression compared to no HRT (n = 16). With E+MPA HRT, there was a further decrease in nuclear p27 and increased Receptor Activator of NF-kappa B Ligand (RANKL) expression compared to E-alone HRT. In luminal breast cancers, E+MPA HRT (n = 6) was also associated with decreased nuclear expression of the cell cycle inhibitor p27 compared to E HRT (n = 6), but was not associated with increased proliferation. These results suggest that p27 mediates progestin-induced proliferation in the normal human breast and that regulation of this proliferative response by E+MPA is lost in breast tumors.

Abstract P5-01-01: Personalized neoadjuvant treatment planning using optical metabolic imaging

Abstract Currently, there are no reliable methods to optimize treatment regimens for individual breast cancer patients. Oncologists choose drug treatments based on expression levels of tumor cell signaling receptors (i.e. HER2, ER, PR) and other factors, and assess whether the treatment is effective after significant time has passed. Unfortunately, over one third of patients exhibit resistance to their initial treatment, increasing their risk of future metastasis and death. Morbidities from sub-optimal drug regimens could be reduced with a personalized drug screen for breast cancer at the time of diagnosis. With the vast number of therapeutic options available to patients (>50 drugs approved with more on the way), a high-throughput screening technology is needed to accurately evaluate how a patient will respond to these options. Here we present Optical Metabolic Imaging (OMI) of tumor-derived organoids as a predictive drug screening platform for individual breast cancer patients. Changes in cell metabolism precede changes in tumor volume and thus present an earlier marker of treatment response. OMI is sensitive to these early changes by exploiting the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), as well as the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox balance, and the fluorescence lifetimes report on the binding activity of these coenzymes. OMI has the unique ability to non-invasively monitor metabolism in living, intact samples on the single-cell level, and can thus quantify heterogeneity in drug response. Changes were quantified at the single-cell level using the OMI Index, a linear combination of the optical redox ratio and the mean NAD(P)H and FAD fluorescence lifetimes. This index was derived using a multivariate analysis of variance and has been shown previously to correlate with treatment response in human cancer cells. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These samples are grown as organoids in a 3D matrix that mimics the natural tumor environment. Organoids were successfully generated from core needle biopsies of untreated breast tumors. These organoids were treated with the patient's prescribed neoadjuvant therapy, and early metabolic changes were quantified using OMI. Organoids grew from a variety of untreated breast tumor subtypes, and early metabolic changes could be resolved at the single-cell level after only 24 hours of treatment in vitro. In parallel, each patient's Residual Cancer Burden (RCB) score was quantified by a surgical pathologist after neoadjuvant treatment and served as gold standard validation of tumor drug response. Results from an early cohort of patients suggest that OMI could be used to predict patient clinical response to therapy. A linear combination of OMI variables measured in vitro in only 48 hours correlated strongly with patient RCB score (Pearson correlation coefficient=0.97, n=5). This methodology could allow oncologists to determine the ideal treatment regimen for their patients at the time of diagnosis. Citation Format: Sharick JT, Walsh AJ, Sanders ME, Kelley MC, Meszoely IM, Hooks MA, Burkard ME, Esbona K, Choudhary A, Skala MC. Personalized neoadjuvant treatment planning using optical metabolic imaging [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-01-01.

Messenger RNA of the Histidine-Rich Glycoprotein in Breast Tumors.

The content of mRNA of the histidine-rich glycoprotein (HRG), a potential marker of malignant neoplasia, which can be used in differential diagnosis of breast tumors, was determined in 110 breast tumor biopsy samples. The presence of HRG mRNA did not depend on the cancer type, on the preoperative treatment or its absence, as well as on the tumor progression stage and the presence of metastases.

The Importance of Immunohistochemistry Techniques in Assessing the Prognosis of Multimodally Treated Breast Cancer

The identification of prognostic factors in the invasive breast cancer is particularly important for the therapeutic approach appropriate to the aggression of neoplasia. Determining the impact of multiple prognostic factors and correlating clinical and immunohistochemical factors allow the treatment to be adjusted so as to avoid the over-treatment of less aggressive neoplasms and to select the optimal multimodal neoadjuvant and adjuvant treatment in cancers with increased aggressiveness. Tissue samples obtained by breast tumor biopsy were studied by classical histopathology methods and by immunohistochemistry techniques, followed by the determination of the histological type, degree of tumor differentiation, status of estrogenic and progesterone receptors, Her2 status. Subsequently, the analysis of the particularities of the prognostic factors associated with the cases with aggressive, metastatic evolution was performed.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-β, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

31P Molecular mechanisms of regulation of proliferation markers of breast cancer in young patients

Background Completetumorregression(stageIV)isaccompaniedbythehighestrates ofoverallsurvival(OS):5-year,and15-yearsurvivalinpatientsofthisgroupis92.3% and83.1%,respectively.Incontrast,inlowpathomorphismOSforthesameperiodwas 42.3% and 28.7%, respectively. Methods We investigated the tissue samples taken during trephine biopsies of breast tumors in 54 women with breast cancer with the mean age of 28-35 years with histologically verified malignant tumors of the mammary glands. Spectrum of markers in tissue samples comprises determination of the degree of tumor malignancy, the level of Ki-67 expression, mitotic index, and cell fraction in S-phase. Results We found that the most sensitive to cytotoxic drugs were proliferating pool of cellsandcellsinS-phase.The3rddegreeofhistologicalmalignancy,Ki67expression level>30%associatedwithagreaterprobabilityofcompleteregressionoftumorsin response to chemotherapy. Poorly differentiated cancers are more sensitive to chemotherapy; the frequency of complete morphological effects is directly proportional to the degree of anaplasia: at tumor grades 1 and 2 the complete pathologic response rate is only 10%, but in highly aggressive tumors when chemotherapy is used it increases up to 90%. In our study, estrogen receptor status appeared an independent factor which predicted complete pathomorphological response and favorable remote results. When comparing the significance of proliferation markers, the significantly predictive value were indicators such as cell fraction in S-phase, mitotic index, the level of Ki 67. The most important value for complete pathomorphism was mitotic index: when its level wasmorethan17to3.3mm2pCRratereached50%,andatthelevelswhichwerebelow the threshold - just 7%. Conclusions Thus, when mitotic index decreased significantly even at large size of residual tumor, the prognosis of the disease is relatively favorable. In cases where high mitotic index and Ki 67 index are associated with gene p53 mutation, the likelihood of significant effect of neoadjuvant chemotherapy is very low, and remote results of treatment are significantly worse. Legal entity responsible for the study N/A Funding N/A Disclosure All authors have declared no conflicts of interest.

Emphysematous Cholecystitis in a Patient without Known Risk Factors: A Case Report

A 64-year-old woman presented with acute right upper abdominal pain and fever. Abdominal computed tomography revealed emphysematous cholecystitis, pneumobilia and a liver tumor. A left breast tumor was also noted upon physical examination. The patient underwent catheter drainage of the gallbladder, but her symptoms were barely relieved; therefore, a cholecystectomy, liver tumor resection and a biopsy of the left breast tumor were performed later. Pathologic analysis revealed emphysematous cholecystitis with gangrenous change of the gallbladder and an invasive ductal carcinoma of the left breast with hepatic metastasis. Our patient has no common risk factors of emphysematous cholecystitis, such as diabetes, old age or the male gender. Left breast cancer with liver metastasis may be a predisposing factor.

Abstract 352: HER2 protein isoform heterogeneity investigated by single-cell western blotting

Abstract Accounting for ∼20% of breast cancers, half of metastatic HER2-positive breast cancer patients treated with trastuzumab plus chemotherapy will experience progression of disease within one year. Trastuzumab treatment, though associated with improvement in median overall survival, still sees drug resistance as an unmet challenge. Differential response to therapy may arise from tumor heterogeneity. Further complicating the situation is the fact that HER2/erbB2 biology is complex. HER2 is not simply one full-length protein (p185) overexpressed on tumor cell surfaces, but also includes truncated isoforms, t-erbB2 (e.g., 95 and 110 kDa isoforms) lacking the extracellular domain targeted by trastuzumab (and pertuzumab and T-DM1). The t-erbB2 isoforms are implicated in tumorigenesis, metastasis, and therapeutic response. Given existing pathology and life sciences tools, the high heterogeneity and need for single cell resolution presents a tremendous challenge. We introduce here a novel microfluidic device to quantitatively assess HER2 protein isoforms in heterogeneous tumor biopsies with single-cell resolution. The single-cell western blot (scWB) provides both the sensitivity and specificity needed to detect t-erbB2 isoforms owing to use of a single-cell electrophoretic protein separation followed by immunoprobing. The assay further provides enough sensitivity to obviate the need for cell pooling, which obscures cell-to-cell heterogeneity. We assay ∼1000's of single cells on one microscope slide in a 4-6 hr workflow. First, we validated that the scWB can separate full length (p185) from truncated (p95) HER2 in single p185- and p95-genetically engineered CHO cells. The p95 was resolved from p185 protein in a short ∼1 mm separation lane (resolution = 0.97±0.03, n>500 cells). Next, we assessed a HER2-positive breast cancer cell line (BT474) and found <3% t-erbB2 expressing cells. To further investigate the effect of t-erbB2 in HER2-related signaling, we examined phospho-proteins including p-HER2, p-HER3, p-ERK Thr187/Tyr185, p-ERK Thr202/Tyr204, p-rs6 from each t-erbB2 expressing BT474 cell. Protein activation in t-erbB2 expressing cells was similar to cells expressing full length HER2. This first-in-kind study demonstrates a powerful single-cell resolution multiplexing capability including for phospho-proteins. We are now applying the novel scWB to measure t-erbB2 in HER2-positive and triple positive (ER/PR/HER2) breast tumor biopsies and will report our results at AACR. Taken together, the microfluidic single-cell resolution western provides a sensitive, quantitative, and multiplexed tool to assay clinically-relevant oncoprotein isoforms that are currently difficult or impossible to assay. Correlating isoform expression ratios with clinical outcomes may confer a more comprehensive understanding of drug resistance responses derived from oncoprotein isoform profiles within heterogeneous cancer cell populations. Citation Format: Ginny (Chi-Chih) Kang, Toby M. Ward, Jessica Bockhorn, Mark D. Pegram, Amy E. Herr. HER2 protein isoform heterogeneity investigated by single-cell western blotting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 352.

Abstract 3023: Cytoplasmic sequestration and autophagic degradation of ErbB receptors in HER2-driven cancer cells by small molecule Sigma1 modulators

Abstract Epidermal growth factor receptors (EGFR/ErbB) drive cell growth, survival, metastasis, and resistance in a range of cancers. Heterodimerization of ErbB1/EGFR, ErbB2/HER2, and ErbB3/HER3 drives aggressive tumor growth through hyperactivation of cancer cell survival and growth signaling pathways. The increased protein production required to sustain these activities renders cancer cells acutely dependent on support factors, such as chaperones and scaffolds, that maintain protein homeostasis. ErbB receptors are integral membrane proteins and as such are synthesized in and transported through the secretory pathway, which comprises the endoplasmic reticulum (ER), Golgi, and associated compartments and vesicles. Nascent ErbB receptors are stabilized and chaperoned through this pathway, in part, by heat shock protein 90 family members, HSP90 and GRP94. Sigma1 (also known as sigma1 receptor) is a unique integral membrane protein found primarily in the ER. Emerging lines of evidence suggest that Sigma1 may function as a chaperone or possibly a scaffolding protein. We find that the levels of Sigma1 protein are elevated and aberrantly distributed in HER2-amplified breast tumor biopsies compared to benign breast tissue. These data indicate that the status and potentially the physiological role of Sigma1 are altered in malignancy and that Sigma1 may be a valid drug target in the treatment of HER2-driven breast cancers. Previously, we discovered that certain selective small molecule modulators of Sigma1 could be used to induce the unfolded protein response (UPR) and autophagy in a panel of cancer cell lines. Here, we demonstrate that these responses to Sigma1 modulators can be exploited to alter the trafficking, stability, and thus signaling of ErbB receptors in cancer cells. In vivo, Sigma1 modulators suppress the growth of xenografted HER2-amplified breast tumors. In the tumors, as well as in vitro cell culture, ErbB1-3 all are eliminated in response to treatment with prototypic small molecule Sigma1 modulators. This corresponds with suppression of downstream PI3K/Akt signaling and with induction of UPR and autophagy. Using high resolution microscopy and organelle fractionation techniques, we confirmed that the Sigma1 modulators induce cytoplasmic sequestration and subsequent degradation of ErbB receptors in ubiquitin-enriched autophagosomes. This process is blocked by cotreatment with autolysosome inhibitor, bafilomycin A1, suggesting that autophagy is the primary mechanism of Sigma1 modulator induced ErbB receptor degradation. Altogether, these data suggest that Sigma1 is a unique, ligand-operated scaffolding protein that contributes to the trafficking and stability of ErbB receptors in HER2-driven cancer cells. Furthermore, these data suggest that Sigma1 is a druggable component of the protein homeostasis regulatory apparatus of cancer cells. Citation Format: Christina M. Maher, Jane Y. Tong, Charles G. Longen, Mercedes I. Lioni, Jeffrey D. Thomas, Xing Tan, Logan Tyler, Fernando U. Garcia, Felix J. Kim. Cytoplasmic sequestration and autophagic degradation of ErbB receptors in HER2-driven cancer cells by small molecule Sigma1 modulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3023.

Feasibility analysis of sentinel lymph node biopsy in patients with breast cancer after local lumpectomy

To explore the feasibility, safety and clinical application value of sentinel lymph node biopsy(SLNB)in patients with breast cancer after local lumpectomy. Clinical data of 195 patients who previously received local lumpectomy from January 2005 to April 2015 were retrospectively analyzed. All the patients with pathologic stage T1-2N0M0 (T1-2N0M0) breast cancer underwent SLNB. Methylene blue, carbon nanoparticles suspension, technetium-99m-labeled dextran, or in combination were used in the SLNB. The interval from lumpectomy to SLNB was 1-91 days(mean, 18.3 days)and the maximum diameter of tumors before first operation was 0.2-4.5 cm (mean, 1.8 cm). The sentinel lymph node was successfully found in all the cases and the detection rate was 100%. 42 patients received axillary lymph node dissection (ALND), 19 patients had pathologically positive sentinel lymph node, with an accuracy rate of 97.6%, sensitivity of 95.0%, false negative rate of 5.0%, and specificity of 100%, and the false positive rate was 0. Logistic regression analysis suggested that the age of patients was significantly associated with sentinel lymph node metastasis after local lumpectomy. For early breast cancer and after breast tumor biopsy, the influence of local lumpectomy on detection rate and accuracy of sentinel lymph node is not significant. Sentinel lymph node biopsy with appropriately chosen tracing technique may still provide a high detection rate and accuracy.

Magnetic Fluorescent Nanoformulation for Intracellular Drug Delivery to Human Breast Cancer, Primary Tumors, and Tumor Biopsies: Beyond Targeting Expectations.

We report the development of a chemotherapeutic nanoformulation made of polyvinylpyrrolidone-stabilized magnetofluorescent nanoparticles (Fl-PMNPs) loaded with anticancer drugs as a promising drug carrier homing to human breast cancer cells, primary tumors, and solid tumors. First, nanoparticle uptake and cell death were evaluated in three types of human breast cells: two metastatic cancerous MCF-7 and MDA-MB-231 cells and nontumorigenic MCF-10A cells. While Fl-PMNPs were not toxic to cells even at the highest concentrations used, Dox-loaded Fl-PMNPs showed significant potency, effectively killing the different breast cancer cells, albeit at different affinities. Interestingly and superior to free Dox, Dox-loaded Fl-PMNPs were found to be more effective in killing the metastatic cells (2- to 3-fold enhanced cytotoxicities for MDA-MB-231 compared to MCF-7), compared to the normal noncancerous MCF-10A cells (up to 8-fold), suggesting huge potentials as selective anticancer agents. Electron and live confocal microscopy imaging mechanistically confirmed that the nanoparticles were successfully endocytosed and packaged into vesicles inside the cytoplasm, where Dox is released and then translocated to the nucleus exerting its cytotoxic action and causing apoptotic cell death. Furthermore, commendable and enhanced penetration in 3D multilayered primary tumor cells derived from primary lesions as well as in patient breast tumor biopsies was observed, killing the tumor cells inside. The designed nanocarriers described here can potentially open new opportunities for breast cancer patients, especially in theranostic imaging and hyperthermia. While many prior studies have focused on targeting ligands to specific receptors to improve efficacies, we discovered that even with passive-targeted tailored delivery system enhanced toxic responses can be attained.

Burkitt’s Lymphoma with Bilateral Cavernous Sinus Involvement: A Case Report

Burkitt’s lymphoma is the commonest childhood malignancy in tropical Africa and is rare outside of Africa. Cavernous sinus lymphoma is a rare occurence and Burkitt’slymphoma of the cavernous sinus is uncommon. We review the case of a 52-year-old non-african and immunocompetent woman who developed over a period of 2 weeks an ocular pain, diplopia and ptosis. Physical examination revealed breast tumors. CT scan and MRI disclosed a bilateral cavernous sinus mass and work-up of the tumor showed additionnal tumor within the pelvis. Biopsy of breast tumor showed findings consistent with Burkitt’s Lymphoma and there was positivity to Epstein-Barr EBER-1. Chemotherapy led to complete and asymptomatic remission of the cavernous sinuses lesions. The disease had a highly aggressive course and the patient died 11 months after diagnosis. We discuss the MRI findings and the differential diagnosis of cavernous sinuses lesions.The case herein reported emphasizes this extremely rare location of Burkitt’s lymphoma, the MR findings and the need of considering the diagnosis of Burkitt’s Lymphoma in cavernous sinuses lesions.

Abstract P1-01-15: Breast cancer biomarkers: Global tumor biomarker quality assurance study of intratumor heterogeneity

Abstract Introduction Biomarkers (BM) are often performed on core biopsies(CB) of breast tumors for treatment purposes, yet comprehensive information on BM heterogeneity is lacking. This quality assurance study is to survey BM intra-tumor heterogeneity (ITH) by comparing BM from CB and the examination of entire breast tumors from surgical excisions (SE). This interim report supplies data on 45/100 collected cases to date. Methods: Whole tumor cases, 3.0cm or less were collected in years 2013-2014, excluding neoadjuvant cases and were examined with CB by two pathologists. All tissues were processed using 2013 ASCO/CAP guidelines. Immunohistochemistry (IHC) for ER/PR/Her2/Ki67 was performed on breast core biopsies and each block of a tumor surgical excision (1-13 blocks per case). IHC was performed using the Ultra Ventana-Roche device with FDA IVD clones ER (SP1), PR(1E2) Her2(4B5) Ki67(30-9). Whole slides were semi-quantitated for ER/PR using HScore (0-300), Her2 was interpreted with 2013 ASCO/CAP guidelines and Ki67 proliferation index (PI) as estimated whole slide percent of tumor cell nuclear stain. Mean HScores and PI were calculated for all BM on SE, and CB deviations from the SE mean were calculated for ER/PR/Ki67. ITH of BM was assessed by comparing results among all tissue blocks for each case. Her2 results were descriptive. Eight cases lacked CB Ki67 PI. Results There were no cases in which categorical hormone receptors results (positive/negative) differed between CB and SE. 2/45 (4%) cases were Her2 positive, and 13/45(28%) were equivocal by IHC with one case amplified by in situ hybridization. Comparing ER between CB/SE , 3/45 differed in HScores by 30% or more. 10/45 SE ER had ITH of 25% or more among different tissue blocks. 14/45 cases had CB/SE PR difference in HScore of 30% or more. 18/45 cases had SE PR with ITH HScore of 25% or more between tissue blocks. Ki67 PI differed between CB/SE by more than 25% in 18/37 cases, with ITH among tissue blocks of more than 25% in 23/37 cases. The variability of CB-SE results and ITH among tissue blocks are summarized in table 1. Table 1 Summary of ResultsCB-SE ERITH ERCB-SE PRITH PRCB- SE PIITH PI>30%>25%>30%>25%>25%>25%3/4510/45 (22%)14/45 (31%)18/45 (40%)18/37(49%)23/37(62%)CB-SE: core biopsy vs surgical excision; ER PR as H Score Conclusions In this interim report of 45/100 cases, (1) ITH of 25% or more occurred for ER content in 22 % of cases (2) similar ITH exists for PR content in 40% of cases. (3) Ki67 PI tissue block ITH was expressed with variation of 25% or more in 23/37 cases. (4) While this degree of variability of BM may not affect treatment decisions, the tissues show sufficient BM variability that should stimulate concern for accuracy of results of prognostic gene expression profiles and genomic testing of breast tumors. Citation Format: Dabbs DJ, Clark B. Breast cancer biomarkers: Global tumor biomarker quality assurance study of intratumor heterogeneity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-15.

Abstract P3-05-14: A neoadjuvant window trial of endocrine response in women with invasive lobular carcinoma

Abstract Background: Patients with invasive lobular carcinoma (ILC) would be expected to have favorable outcomes compared to patients with invasive ductal carcinoma (IDC) given that ILC is more often hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative, of lower grade, and displays decreased proliferation markers. Based on our preclinical studies showing differential hormone response in HR+ ILC vs. IDC and on recent studies suggesting differences in endocrine treatment response between patients with ILC vs. IDC, we designed a biomarker-driven, neoadjuvant window trial for newly diagnosed women with HR+, HER2-negative ILC. We hypothesize that Ki67 will be reduced by 85% in the fulvestrant arm compared with 60% and 75% reduction in the tamoxifen and anastrozole arms, respectively, and that Ki67 reduction will correlate with alterations in expression of ER and ER-regulated genes. Differential Ki67 effect will serve as a surrogate for outcome of patients with ILC on endocrine therapy. Trial Design: This multicenter study (NCT02206984) will enroll 150 women with HR+ and HER2-negative ILC. A mandatory research breast tumor biopsy will be performed at baseline. Fifty patients will be randomized to each of three open-label treatment arms for 21 days: fulvestrant (two 250 mg IM injections on both day 1 and day 14), anastrozole (1mg orally daily), or tamoxifen (20 mg orally daily). Biomarkers of response will be assessed on baseline and post-treatment tumor tissue. Patients will proceed to definitive surgery on day 21 after study drug exposure, or they will undergo a second research breast core biopsy if further neoadjuvant treatment is planned. Eligibility Criteria: Eligible patients include postmenopausal women with newly diagnosed, HR+, HER2-negative ILC (excluding pleomorphic subtype) measuring ≥ 1cm, with adequate organ function, ECOG PS ≥ 2, and agreeable to baseline research breast tumor biopsy. Specific Aims: The primary endpoint is percent change from baseline to post-treatment Ki67 values in ILC tissue after 21 days of endocrine treatment. Comparisons across study arms will be made using a general linear model adjusting for institutional effect, with 80% power estimated for pairwise comparisons of log2(% staining) between treatment arms, allowing for 10% attrition. Secondary endpoints include post-therapy Ki67, and change in ER and PR protein expression by IHC. Finally, planned correlative studies include evaluation of gene expression, epigenetic markers, and DNA sequence variants in ILC tissues in an effort to identify biomarkers of endocrine response and putative drivers of endocrine resistance in ILC. Target Accrual: This study will be open to enrollment by August 2015 at the University of Pittsburgh. Additional sites will be opened through the Translational Breast Cancer Research Consortium (TBCRC). We anticipate an accrual rate of 8 patients per month. (Funding from Susan G. Komen® and AstraZeneca). Citation Format: Jankowitz RC, McAuliffe PF, Sikora MJ, Butler L, Ahrendt G, Johnson R, Diego E, Bonaventura M, Puhalla S, Lembersky B, Clark B, Brufsky A, Kurland BF, Davidson NE, Dabbs DJ, Oesterreich S. A neoadjuvant window trial of endocrine response in women with invasive lobular carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-14.

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

Abstract 1063: Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues

Abstract Introduction: DNA methylation variability regions (MVRs) across the estrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumor biopsies and exfoliated epithelial cells from expressed breast milk (EBM) to determine their significance for ESR1 transcription. Methods: DNA methylation levels in eight MVRs across ESR1 were assessed in pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with the demethylating agent, decitabine (DAC, 1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumors (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. Results: We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumor biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumor cell lines. Conclusion: Intragenic methylation levels correlated with ESR1 expression in vitro, but with markedly different methylation patterns between homogenous cell populations and heterogeneous tumour biopsies. DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter along with reduced levels of intragenic DNA methylation. These findings emphasize the need for care when choosing tissue types for epigenetic research and interpreting results from heterogenous tissue. Note: This abstract was not presented at the meeting. Citation Format: NATALIE S. SHENKER, Kirsty J. Flower, Charlotte Wilhelm-Benartzi, Wei Dai, Emma Bell, Mona El Bahrawy, Gillian Weaver, James M. Flanagan, Robert Brown. Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2015-1063

Abstract 3082: miR-155-5p expression is associated with response to neoadjuvant paclitaxel treatment of locally advanced breast cancer

Abstract Introduction Resistance to chemotherapy remains a prominent obstacle to cure in cancer. We have previously shown that genetic defects in key pathways involved in processes like apoptosis, cell-cycle arrest and senescence may be involved in resistance to chemotherapy in vivo. MicroRNAs (miRNAs) are known to be crucial regulators of gene expression, and to potentially affect the response to drugs, including chemotherapy. Here, we aimed to explore the potential role of miRNAs in mechanisms of breast cancer chemoresistance in-vivo, by analyzing miRNA expression in breast cancer patients with recorded responses to chemotherapy. Patient materials and Methods Breast tumour biopsies were collected before chemotherapy in a prospective clinical study, where patients were randomized to receive epirubicin or paclitaxel monotherapy (n = 223). Information about the response to therapy, as defined by the UICC criteria, in addition to long-term outcome, was available for all patients. The global miRNA expression levels were analyzed in 50 breast tumour biopsies by deep sequencing. The patients were selected based on their response to first-line chemotherapy. To verify the results obtained by miRNA sequencing, selected miRNAs were quantified in all patients by qPCR. Results No global miRNA patterns were observed when performing unsupervised clustering of miRNA sequencing data. Among samples from paclitaxel-treated patients, we identified three differentially expressed miRNAs between response groups; the expression of miR-155 was significantly lower (p = 0.005) in patients with poor response to paclitaxel, and the expression of both miR-181a and miR-181b were significantly higher (p = 0.003 and 0.012, respectively) in patients with poor response to paclitaxel. qPCR data validated the initial association identified between high miR-155-5p expression levels and response to paclitaxel treatment. Intriguingly though, high levels of miR-155 were also associated with a poor prognosis in the study population as a whole (p = 0.012), as well as with the presence of mutations in TP53 (p < 0.001). Conclusion Here, we show that breast cancer patients responding to paclitaxel treatment have higher expression levels of miR-155-5p. Also, patients with elevated levels of miR-155-5p expression have a poor prognosis and a higher frequency of TP53 mutations. Citation Format: Anne Hege Straume, Einar Birkeland, Stian Knappskog, Per Eystein Lønning. miR-155-5p expression is associated with response to neoadjuvant paclitaxel treatment of locally advanced breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2015-3082